November 2012
Volume 53, Issue 12
Biochemistry and Molecular Biology  |   November 2012
In Vitro Effects of Bevacizumab Treatment on Newborn Rat Retinal Cell Proliferation, Death, and Differentiation
Author Affiliations & Notes
  • Nádia C. O. Miguel
    From the Programa de Biologia Celular e do Desenvolvimento, Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil; the
    Laboratory for Investigation in Ophthalmology (LIM-33), University of São Paulo Medical School São Paulo, Brazil; the
  • Monique Matsuda
    Laboratory for Investigation in Ophthalmology (LIM-33), University of São Paulo Medical School São Paulo, Brazil; the
  • André Luís F. Portes
    Laboratory for Investigation in Ophthalmology (LIM-33), University of São Paulo Medical School São Paulo, Brazil; the
  • Silvana Allodi
    Programa de Neurobiologia, and the
  • Rosália Mendez-Otero
    Programa de Terapia Celular e Bioengenharia, Instituto de Biofisica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil.
  • Thiago Puntar
    From the Programa de Biologia Celular e do Desenvolvimento, Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil; the
  • Alfred Sholl-Franco
    Programa de Neurobiologia, and the
  • Paloma G. Krempel
    Programa de Terapia Celular e Bioengenharia, Instituto de Biofisica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil.
  • Mário L. R. Monteiro
    Laboratory for Investigation in Ophthalmology (LIM-33), University of São Paulo Medical School São Paulo, Brazil; the
  • Corresponding author: Mário L. R. Monteiro, Av. Angélica 1757 conj 61, 01227-200, São Paulo, Brazil;
Investigative Ophthalmology & Visual Science November 2012, Vol.53, 7904-7911. doi:10.1167/iovs.12-10283
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      Nádia C. O. Miguel, Monique Matsuda, André Luís F. Portes, Silvana Allodi, Rosália Mendez-Otero, Thiago Puntar, Alfred Sholl-Franco, Paloma G. Krempel, Mário L. R. Monteiro; In Vitro Effects of Bevacizumab Treatment on Newborn Rat Retinal Cell Proliferation, Death, and Differentiation. Invest. Ophthalmol. Vis. Sci. 2012;53(12):7904-7911. doi: 10.1167/iovs.12-10283.

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      © ARVO (1962-2015); The Authors (2016-present)

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Purpose.: Vascular endothelial growth factor (VEGF) is an important signal protein in vertebrate nervous development, promoting neurogenesis, neuronal patterning, and glial cell growth. Bevacizumab, an anti-VEGF agent, has been extensively used for controlling pathological retinal neovascularization in adult and newborn patients, although its effect on the developing retina remains largely unknown. The purpose of this study was to investigate the effect of bevacizumab on cell death, proliferation, and differentiation in newborn rat retina.

Methods.: Retinal explants of sixty 2-day-old Lister hooded rats were obtained after eye enucleation and maintained in culture media with or without bevacizumab for 2 days. Immunohistochemical staining was assessed against proliferating cell nuclear antigen (PCNA, to detect cell proliferation); caspase-3 and beclin-1 (to investigate cell death); and vimentin and glial fibrillary acidic protein (GFAP, markers of glial cells). Gene expressions were quantified by real-time reverse-transcription polymerase chain reaction. Results from treatment and control groups were compared.

Results.: No significant difference in the staining intensity (on immunohistochemistry) of PCNA, caspase-3, beclin-1, and GFAP, or in the levels of PCNA, caspase-3, beclin-1, and vimentin mRNA was observed between the groups. However, a significant increase in vimentin levels and a significant decrease in GFAP mRNA expression were observed in bevacizumab-treated retinal explants compared with controls.

Conclusions.: Bevacizumab did not affect cell death or proliferation in early developing rat retina but appeared to interfere with glial cell maturation by increasing vimentin levels and downregulating GFAP gene expression. Thus, we suggest anti-VEGF agents be used with caution in developing retinal tissue.

Vascular endothelial growth factor (VEGF) is a glycoprotein that plays an important regulatory role in vascular growth in several vertebrate organ systems during development and in adulthood, influencing vessel remodeling, stabilization, and differentiation. 13 Its crucial role in angiogenesis accounts for the importance VEGF plays in early vascular differentiation, wound healing, and in pathological processes such as tumor growth and retinopathies. 46 The study of the role of VEGF in the pathogenesis of several pathological processes has led to the development of specific anti-VEGF agents such as bevacizumab, a humanized monoclonal antibody against all isoforms of VEGF. 7 Bevacizumab has been approved by the Food and Drug Administration for treatment of metastatic colorectal cancer and is used worldwide in the treatment of several chronic vascular eye diseases in adults, such as diabetic retinopathy, retinal vein occlusion, and senile macular degeneration. 710 More recently, bevacizumab has also been introduced as a clinical tool in the treatment of hyperoxia-induced vaso-obliteration followed by neovascular formation in developing eyes of premature infants with retinopathy of prematurity (ROP). 11,12  
In addition to stimulating normal and pathological angiogenesis, VEGF plays a critical role, both neurotrophic and instructive, in neurogenesis. 13,14 Developmental studies have shown that angiogenesis and neural cell differentiation and maturation are almost synchronous in the retina, as in the rest of the nervous system. 15 The differentiation of neuronal and glial cell precursors is stimulated by several growth factors, including basic fibroblast growth factor, insulin-like growth factor-1, transforming growth factor-β, and VEGF. 15,16  
Several studies have evaluated the effects of anti-VEGF agents on retinal tissue when used in the treatment of retinal disease. While some researchers have reported bevacizumab use to be associated with ultrastructural abnormalities in the retina of rabbits, mice and primates, 1719 most have failed to detect any retinal toxicity in the adult retina. 20,21 Only two previous studies have evaluated the effect of anti-VEGF agents on the developing retina in vivo. 22,23 Zayit-Soudry et al. 22 evaluated the effect of intravitreal administration of bevacizumab on the developing retina of 11- or 25-day-old rabbits, but found no electroretinographic or morphological abnormalities; nevertheless, no quantitative analysis of cell death, proliferation, or reactive gliosis were performed. On the other hand, using quantitative immunohistochemistry, Fusco et al. 23 observed more proliferating cells, greater glial cell reactivity, and fewer cells undergoing programmed cell death in the retinas of 28-day-old rabbits injected intravitreously with bevacizumab than in nontreated retinas. 23 However, while the effect was most likely the result of the pharmacological action of bevacizumab, since a sham injection in the fellow eye was not delivered, a contributory effect of the inflammation caused by the trauma of intraocular injection could not be ruled out, pointing out the need for further studies to clarify this issue. 
Therefore, the purpose of this paper was to evaluate the influence of in vitro anti-VEGF bevacizumab treatment upon cell death, differentiation, and proliferation in developing rat retinal explants maintained in organotypic culture. 
Materials and Methods
All procedures were performed in accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals, as described in the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research, and by the National Institutes of Health, and approved by the ethics committees of animal research of our institutions. 
Basal medium of Eagle (BME), HEPES, penicillin G, streptomycin sulfate, and L-glutamine were obtained from Sigma-Aldrich (St. Louis, MO). Bevacizumab was from Genentech, Inc. (Avastin; San Francisco, CA). The RNA extraction kit (RNeasy Mini Kit) and the PCR kit (Rotor Gene SYBR Green PCR Kit) were obtained from Qiagen (São Paulo, Brazil). The reverse transcriptase kit (SuperScript II Reverse Transcriptase Kit) was obtained from Invitrogen (Carlsbad, CA). Primary mouse antibodies for antiproliferating cell nuclear antigen (PCNA) and antivimentin were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), Primary antibody anti-beclin-1 and antiglial fibrillary acidic protein (GFAP) were from Chemicon International, Inc. (Temecula, CA). Primary antibody anti-caspase-3 was obtained from Merck Millipore (Life Science division of Merck KGaA, Germany). Fluorescent secondary antibodies, anti-rabbit IgG CY-3 conjugate and anti-mouse IgG CY-3 conjugate, were from Sigma-Aldrich. All other reagents were of analytical grade. 
Sixty newborn 10-g Lister hooded rats were used. The matrices were kept under a 12/12-hour light/dark cycle with free access to water and food while nursing. 
Retinal Tissue Culture
Rat retinal explants were prepared as described elsewhere. 24 Briefly, 2-day-old rats were killed by decapitation, the eyeballs were removed, and the retinas dissected free from scleral tissue and RPE using a calcium- and magnesium-free balanced salt solution (CMF). The retinas were cut into fragments of approximately 1 mm2 in complete BME supplemented with 2 mM L-glutamine, 100 μg/mL streptomycin sulfate, 100 U/mL penicillin G, 20 mM HEPES, and 5% fetal calf serum. The retinal explants were maintained in 25-mL tight-lidded Erlenmeyer flasks and incubated at 37°C, in an atmosphere of 5% CO2 and 95% air in an orbital shaker at 80 to 90 rpm. After dissection, some explants were assigned as controls while the remainder received culture medium, with or without the addition of 0.5 mg/mL bevacizumab. After 48 hours, retinal explants were either fixed in paraformaldehyde for immunohistochemistry or immersed in RNA stabilization agent (RNAlater; Qiagen) for quantitative real-time PCR analysis (qRT-PCR). 
Histological Procedures
The retinal explants were fixed by immersion in 4% paraformaldehyde in phosphate buffer, 0.1 M, pH 7.4, for 1 hour, followed by immersion in a 30% sucrose solution in phosphate buffer. Tissue orientation was verified under a stereoscopic microscope in aluminum chambers filled with optimum cutting temperature compound. Cross-sections that were 10-μm thick were prepared with a cryostat (Leica CM3000; Leica Microsystems, Wetzlar, Germany), mounted on poly-L-lysine-coated slides, and maintained at −20°C until processing. The orientation procedure ensured that sectioning through the tissue was almost exactly orthogonal to the surface of the retina. 
For immunodetection, sections were incubated with 0.1% Triton in PBS, washed once with PBS/Triton, and treated for 60 minutes at room temperature with a blocking solution (10% bovine serum albumin, BSA) diluted in PBS. After blocking, sections were incubated overnight at 4°C with primary and secondary antibodies at the suggested dilution (1:100 for anti-PCNA, anti-beclin-1, anti-vimentin and anti-GFAP; 1:10 for anti-caspase 3) in 5% BSA diluted in PBS. Further processing was done by incubation for 2 hours at room temperature with specific fluorescent secondary antibodies (anti-rabbit IgG CY-3 conjugate and anti-mouse IgG CY-3 conjugate) diluted in 1% PBS-BSA. After the reactions, preparations using fluorescent-conjugated secondary antibodies were mounted in mounting medium (Fluoromount Aqueous Mounting Medium; Sigma-Aldrich) and conserved at 4°C in a darkened refrigerator. Control sections were processed in the absence of primary antibodies (data not shown). 
The sections were observed, first under a conventional fluorescence microscope (Zeiss Axioskop 2 Plus; Carl Zeiss, Baltimore, MD), then under a confocal microscope (Leica TCS-SP5; Leica Microsystems) at 543-nm wavelength of excitation for the CY-3 fluorochrome. All images were acquired at a resolution of 1024 × 1024 pixels, using 60% laser transmittance, 0.5-μm optical slices, and 2-minute scanning-time in the same value of the detector gain. Negative and positive controls for the reactions were prepared (data not shown). Sections containing the central or peripheral retinal areas from treated and control eyes were evaluated both under the light microscope (63× objective) and the confocal microscope. Five fields of each section of the retina (25 fields of treated and control retinas) were chosen at random. 
For quantitative RT-PCR, total RNA was isolated from rat retinal explants using an RNA extraction kit (Qiagen). Subsequently, cDNAs were generated from 2 μg of total RNA using a reverse transcriptase kit (Invitrogen). The resulting cDNA was subjected to a 40-cycle PCR amplification using the manufacturer's PCR kit (Qiagen) protocol, quantifying PCNA, caspase-3, beclin-1, vimentin, and GFAP transcripts. 
For each gene analysis, three replicates of the ARBP gene were run. ARBP was used as endogenous reference gene as it did not exhibit significant expression abnormalities in any group (data not shown). The standard curves for each gene had adequate slopes, indicating the efficiency of amplification for PCNA, caspase-3, beclin-1, vimentin, and GFAP. The relative quantification method (ΔΔCt) was used, with the ratio of target mRNA, normalized with respect to ARBP mRNA and relative to a calibrator sample using high resolution melting analysis software (Rotor-Gene ScreenClust HRM; Qiagen). 
Statistical Analysis
The results from the two groups (control and treatment) were expressed as mean ± standard error of mean (SEM) and compared using either Student's t-test or the Mann-Whitney U-test, depending on adherence to the normality assumption. The level of statistical significance was set at 5% (P < 0.05). 
In retinas of postnatal vertebrates, such as those used in the present study, the cell population doubling time is 18 hours in vivo, as demonstrated elsewhere. 25 Our retinal explants displayed adequate histological organization characterized by nuclear layers alternating with plexiform layers. Matching results from previous studies, 26 within the period of culture (48 hours) no noticeable changes occurred in either general structure or lamination of the retinal tissue as compared to the retina in situ (data not shown).The maintenance of an adequate histological organization in our organotypic cultures is a strong indicator of the validity of the study model. 
Figure 1 displays immunohistochemical findings for the two groups (treated and untreated) with regard to PCNA, caspase-3, beclin-1, vimentin, and GFAP. Figure 2 shows quantitative immunohistochemical findings, with bar graphs for each group. No significant difference in labeling for PCNA, caspase-3, beclin-1, and GFAP was observed between the groups. However, vimentin antibody levels were significantly higher in bevacizumab-treated retinas (0.24 ± 0.007) than in controls (0.11 ± 0.055; P = 0.016). 
Figure 1. 
Confocal laser scanning photomicrographs of bevacizumab-treated and control retinal explants stained immunohistochemically. (A) PCNA. (B) Caspase. (C) Beclin-1. (D) Vimentin. (E) GFAP. GCL, ganglion cell layer.
Figure 1. 
Confocal laser scanning photomicrographs of bevacizumab-treated and control retinal explants stained immunohistochemically. (A) PCNA. (B) Caspase. (C) Beclin-1. (D) Vimentin. (E) GFAP. GCL, ganglion cell layer.
Figure 2. 
Bar graphs for bevacizumab-treated and control retinal explants showing the intensity of immunofluorescence labeling antibodies. (A) PCNA. (B) Caspase. (C) Beclin-1. (D) Vimentin. (E) GFAP.
Figure 2. 
Bar graphs for bevacizumab-treated and control retinal explants showing the intensity of immunofluorescence labeling antibodies. (A) PCNA. (B) Caspase. (C) Beclin-1. (D) Vimentin. (E) GFAP.
Figure 3 is a bar graph of the RNA expression for beclin-1, PCNA, vimentin, and GFAP. Treatment with bevacizumab did not affect the mRNA expression for caspase-3, beclin-1, PCNA, and vimentin since similar mRNA levels were observed in both groups. However, the GFAP mRNA expression was significantly decreased in bevacizumab-treated retinas (0.24 ± 0.04) compared with controls (0.87 ± 0.21, P = 0.001; Fig. 3), indicating downregulation of the mRNA for GFAP. 
Figure 3. 
Bar graphs showing the results of mRNA transcripts in bevacizumab-treated and control retinal explants. (A) PCNA. (B) Caspase-3. (C) Beclin-1. (D) Vimentin. (E) GFAP.
Figure 3. 
Bar graphs showing the results of mRNA transcripts in bevacizumab-treated and control retinal explants. (A) PCNA. (B) Caspase-3. (C) Beclin-1. (D) Vimentin. (E) GFAP.
Over the last decade, the recognition of the role of VEGF in a wide range of retinal vascular diseases has led physicians to employ anti-VEGF agents, including bevacizumab, in the intravitreous treatment of a large number of vasoproliferative ocular disorders. 27 Ever since the introduction of bevacizumab as a therapeutic choice, its possible toxicity to the retina has been an issue of great concern and object of intense research. 28,29 Clinical and histological studies testing the effect of bevacizumab on rabbit retinal tissue have failed to show tissue toxicity. 21,30 Nevertheless, most safety studies published to date have evaluated the effect of bevacizumab in adult animals, rather than assessing its safety profile in the developing retina. 17,20,21,31,32 During retinal development, VEGF is expressed by astrocytes in the retinal ganglion cell layer, by Müller cells, and other cells of the inner nuclear layer and by RPE cells. 33,34 It has been suggested that VEGF may act as a neuroprotective and neurotrophic factor for retinal cells, influencing neuronal growth, differentiation, and survival. 13,35 Therefore, further studies are needed to investigate the effect of anti-VEGF drugs on the developing retina. 
Zayit-Soudry et al. 22 investigated the effect of the drug injected in 11-day-old and 25-day-old rabbits but found no retinal function abnormalities in the electroretinograms and visual-evoked potentials of bevacizumab-injected eyes compared with contralateral control eyes. No structural retinal damage and no increase in GFAP immunoreactivity was observed in treated eyes compared with controls. However, no quantitative analyses of cell death, proliferation, and gliosis were performed. 22 On the other hand, Fusco et al. 23 evaluated juvenile rabbits treated with bevacizumab intravitreously in one eye while the contralateral eye was assigned as control. Immunohistochemistry revealed fewer cells undergoing programmed cell death, more proliferating cells, and increased reactivity of glial cells in bevacizumab-treated eyes compared with controls. These findings suggest that VEGF could play an important role in the control of retinal cell differentiation and death. The observation of gliosis also suggests that the intravitreal use of this anti-VEGF agent is not completely risk-free. Nevertheless, a possible influence of the injection procedure could not be ruled out. 23  
In the current study, we moved one step further in the analysis by investigating the effect of bevacizumab using an in vitro–organotypic culture of developing retina. To accomplish this, we used explants of developing rat retina, which provide an excellent model to study mechanisms of proliferation, cell death, and differentiation in a complex and highly structured tissue, provided the spatial distribution of the various cell types, the neurochemical circuitry, and the extracellular matrix microenvironment are preserved. 36 Explants of newborn Lister hooded rat retina, as used in this study, are composed of two major cellular strata separated by an emerging layer of processes. The innermost cellular stratum represents the ganglion cell layer, 37 while the outer cellular stratum consists mostly of neuroblasts. 25  
In our study, neither immunohistochemistry nor RT-PCR revealed any differences in cell proliferation, apoptosis, and autophagy in bevacizumab-treated retinas compared with controls. Furthermore, no difference in gene expression for PCNA, caspase-3, and beclin-1 was found (Fig. 3). The results indicate that, at the stage of development evaluated in our study (48 hours postnatal), a single dose of bevacizumab probably does not interfere with retinal cell death or proliferation, in contrast with our previous findings for juvenile rabbits. However, the present findings do not rule out interference at a later stage of development. Indeed, our previous experiments with 28-day-old rabbits centered on a development phase entirely different from the postnatal stage (2 to 4 days) evaluated in the present study. 
However, the finding of a significant increase in vimentin filament in eyes treated with bevacizumab indicates that the substance does in fact interfere with the maturation of retinal glial cells. Vimentin is expressed in processes of Müller glial cells and in retinal astrocytes from the early stages of postnatal retinal development into adulthood. 38 Shaw et al. 38 studied rat retina structure between embryonic day 14 and adult age based on the expression of antibodies, including those specific for vimentin and GFAP. Vimentin could be detected in radial processes throughout the retina at all stages studied. These processes are believed to correspond to ventriculocytes in the developing retina and to Müller cells in the mature retina. 38 The two filaments are observed in coexpression from the embryonic stage to adult age, although, as shown by DeGuevara et al., 39 vimentin is expressed earlier in retinal development than GFAP, particularly in Müller cells. The significant increase in vimentin filament and the downregulation of GFAP gene expression observed in the current study suggests that bevacizumab causes a delay in the maturation of rat glial cells. The high levels of vimentin, despite the absence of upregulation of its regulating gene, are possibly the result of accumulation (or slower degradation) of the filament in the tissue. The normal levels of GFAP despite the downregulation of its gene also points to an interference of bevacizumab in the maturation of rat retina. 
Previous reports have considered, but not confirmed, the possibility that anti-VEGF antibodies might induce Müller cell gliosis. 14,40 Guo et al. 14 evaluated the effect of bevacizumab on cultured rat retinal Müller glial cells as well as in vivo in adult rats injected intravitreously. The authors concluded that bevacizumab exerts no inhibition of rat retinal Müller glial cells. Iandiev et al. 40 evaluated the effect of bevacizumab on the retina of adult pigs and concluded that a single intravitreal injection of bevacizumab does not induce gliosis. However, both studies evaluated retinal glial cells of adult animals and their findings cannot be extrapolated to the developing retina. The delayed maturation of very early developmental rat retinal glial cells reported in the current study is in agreement with our previous observation of increased levels of GFAP (a Müller cell cytoskeleton protein expressed at a later stage) in the retina of juvenile rabbits submitted to intravitreous bevacizumab injection. 23 Since VEGF is known for its importance to the maturation and differentiation of the nervous system, the observation that anti-VEGF bevacizumab treatment influences Müller cell biology, especially during development and maturation, is not surprising. 
It may be argued that bevacizumab might not be effective in rat retina. Bevacizumab it is a humanized variant of the anti-VEGF-A murine antibody A.4.6.1, originally described as effective against all isoforms of VEGF in the rat. 41 It has been considered as a species-specific antibody having little interaction with murine VEGF-A. 42 However, Bock et al. 43 reported that bevacizumab had the ability to bind to murine VEGF-A in three independent molecular biological assays (Western blot analysis, ELISA, and BIAcore assay). Furthermore, several studies have indicated bevacizumab is effective in the rat based on animal studies investigating its influence on corneal angiogenesis, 4346 retinal and choroidal vessels, 19,47 and rat retinal cell culture. 48 Therefore, although bevacizumab may not have the same binding affinity to VEGF in rats as it does in humans, there is enough evidence to indicate that bevacizumab is active in rat models, as observed in our study. 
Our current results, as well as the findings of previous studies, suggest that the use of this anti-VEGF agent in immature eyes may not be risk-free. While the lack of apoptosis in our results might be viewed as a point in favor of the safety of the drug, the interference in vimentin and GFAP metabolism does indicate a possible interference in early retinal maturation, particularly of Müller glial cells. Since glial cells provide physical scaffold for retinal organization and contribute to the removal and recycling of neurotransmitters, 49 which strongly impacts neurogenesis, disturbance in its differentiation might therefore interfere on other retinal cell development. This concern is addressed in the particular case of preterm babies suffering from ROP, a leading cause of lifelong visual impairment and blindness in premature babies. 50 In these patients, the retina is also under development, and the blockage of VEGF, as well as the potential side effects of anti-VEGF drugs such as bevacizumab, remain unknown. 50 To our knowledge, no clinical trials monitoring preterms receiving local treatment with bevacizumab have been completed, and studies of the long-term effects of this drug on the developing retina are still lacking. 51 Our findings reinforce the suggestion made by others 51 that off-label administration of bevacizumab should be prescribed with caution in such cases. 
It is also important to point out some limitations of our study. Based on previous studies on the maturation of human versus rat retinas using growth curves for rhodopsin as landmark of retinal differentiation, 52 it is very likely that the 2- to 4-day-old rat retinas evaluated in our study are less mature than the retinas of babies with ROP. In fact, most rat experimental models of ROP analyze retinas around postnatal day 12 to 1519 when the rat retinal development is similar to that of premature infants. Therefore, the deleterious effect on vimentin and GFAP expression observed at the age addressed in our study may not apply directly to children with ROP. On the other hand, previous studies have demonstrated that vimentin and GFAP filaments are expressed in rat and human retinas from the embryonic period until the neural retina development is complete. 38,53,54 It is therefore very likely that abnormalities observed in our early developing retinas may also be operating later in retinal development, particularly when bevacizumab is used in human eyes, where its anti-VEGF activity is expected to be much greater than that observed in the rat retina. However, in order to be certain of its potential consequences to the developing retina of premature newborns suffering from retinopathy of prematurity, our findings should be complemented by other investigative approaches. 
In conclusion, our study indicates that bevacizumab interferes with the early differentiation of retinal glial cells in 2-day-old Lister hooded rat explants by increasing vimentin content and downregulating the GFAP gene expression. Therefore, it should be used with caution when treating developing eyes, particularly in premature newborn infants. 
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 Supported by grants from Rio de Janeiro State Foundation for the Advancement of Science (FAPERJ) and Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP, Grant No. 2011/50174-0), São Paulo, Brazil. The authors alone are responsible for the content and writing of the paper.
 Disclosure: N.C.O. Miguel, None; M. Matsuda, None; A.L.F. Portes, None; S. Allodi, None; R. Mendez-Otero, None; T. Puntar, None; A. Sholl-Franco, None; P.G. Krempel, None; M.L.R. Monteiro, None
Figure 1. 
Confocal laser scanning photomicrographs of bevacizumab-treated and control retinal explants stained immunohistochemically. (A) PCNA. (B) Caspase. (C) Beclin-1. (D) Vimentin. (E) GFAP. GCL, ganglion cell layer.
Figure 1. 
Confocal laser scanning photomicrographs of bevacizumab-treated and control retinal explants stained immunohistochemically. (A) PCNA. (B) Caspase. (C) Beclin-1. (D) Vimentin. (E) GFAP. GCL, ganglion cell layer.
Figure 2. 
Bar graphs for bevacizumab-treated and control retinal explants showing the intensity of immunofluorescence labeling antibodies. (A) PCNA. (B) Caspase. (C) Beclin-1. (D) Vimentin. (E) GFAP.
Figure 2. 
Bar graphs for bevacizumab-treated and control retinal explants showing the intensity of immunofluorescence labeling antibodies. (A) PCNA. (B) Caspase. (C) Beclin-1. (D) Vimentin. (E) GFAP.
Figure 3. 
Bar graphs showing the results of mRNA transcripts in bevacizumab-treated and control retinal explants. (A) PCNA. (B) Caspase-3. (C) Beclin-1. (D) Vimentin. (E) GFAP.
Figure 3. 
Bar graphs showing the results of mRNA transcripts in bevacizumab-treated and control retinal explants. (A) PCNA. (B) Caspase-3. (C) Beclin-1. (D) Vimentin. (E) GFAP.

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